This application is based upon and claims the benefit of priority from the prior Japanese Patent Application No. 11-206535, filed Jul. 21, 1999, the entire contents of which are incorporated herein by reference.
The present invention relates to a membrane-bound complement regulatory protein capable of suppressing an initial reaction of a complement pathway which causes a hyperacute rejection in a subject (donated organ) when organs, tissues or cells are transplanted. The present invention also relates to a nucleic acid molecule encoding the membrane-bound complement regulatory protein and a vector having the nucleic acid molecule. The present invention further relates to a cell, a tissue and an organ having the vector introduced therein. Moreover, the present invention relates to a transgenic animal, particularly, a transgenic swine in which a gene of the membrane-bound complement regulatory protein is introduced.
Organ transplantation is an extremely useful radical treatment and completely differs from a conventional symptomatic treatment. Recently, transplantation of organs such as kidney, cornea, liver, and heart has been frequently and widely performed to recover dysfunction or hypofunction of the organs.
The most significant problem which should be overcome in the organ transplantation is a rejection. In the case of kidney transplantation from human to human, which is one of the most widely performed organ transplantation, a hyperacute rejection occurs within several minutes after transplantation in a case where blood types do not match with each other, with the result that severe propagated thrombosis occurs around the transplanted organ. It has been elucidated that the hyperacute rejection at the time of allotransplantation (the donor and recipient belong to the same species) is caused by activating a complement pathway by binding C1 (a first component of complement) to an immune complex, which consists of a blood type determinant glycogenic antigen of a graft and an antibody against the blood type determinant glycogenic antigen inherently present as a natural antibody in the recipient. In the case of xenotransplantation (the donor and recipient belongs to different species), the rejection would be more serious since all substances not present in the recipient may come to be possible xenotransplantation antigens. Therefore, the rejection is a big problem of preventing clinical application of the xenotransplantation which enables donation of numerous organs.
As described above, the primary issue to be solved in the organ transplantation is to inhibit the hyperacute rejection occurring immediately after transplantation. Since the hyperacute rejection is caused by activating the complement pathway as previously described, if a suppressive substance for the pathway is introduced into the organ to be transplanted in advance, the hyperacute rejection can be inhibited.
Currently, based on the aforementioned idea, several manufacturers actually put production of transgenic swine into practice by introducing genes of endogenous human membrane-bound complement regulatory proteins (hereinafter referred to as xe2x80x9cmbCRPsxe2x80x9d), such as CD 46 (membrane cofactor protein; MCP), CD55 (decay accelerating factor, DAF) and CD59 (HRF 20), and actually apply to the xenotransplantation. The term xe2x80x9ccomplement regulatory protein (hereinafter referred to an xe2x80x9cCRPxe2x80x9d)xe2x80x9d used herein refers to a protein regulating the complement pathway in a living body. Almost all the CRPs including the aforementioned three proteins have a function of regulating a complement activity. Therefore, if an organ of the transgenic animal having the CRP introduced therein is transplanted to a recipient, the complement pathway of the recipient can be suppressed.
Now, referring to FIG. 1, working points of the mbCRPs, namely, CD46, CD55 and CD59 in the compliment pathway will be explained. At first, the working point of CD46 resides in the reaction represented by a reference numeral (3) in FIG. 1. CD 46 functions as a cofactor of factor I in converting C3b and C4b into inactivated forms. C3b is a molecule playing a pivotal role in the complement pathway. More specifically, C3b plays a role in
{circle around (1)} activating a C3 convertase (YC3bBbP in FIG. 1) of an alternative pathway,
{circle around (2)} converting C3 convertases (YC4b2a and YC3bBbP in FIG. 1) into a C5 convertase (YC4b2a3b),
{circle around (3)} mediating binding to a complement receptor type I, CR1, of a blood cell.
On the other hand, the working point of CDS5 resides in the reaction represented by a reference numeral (2) in FIG. 1. More specifically, CD 55 promotes dissociation of C2a* from the C3 convertase C4b2a in the classical complement pathway and simultaneously promotes dissociation of Bb from the C3 convertase YC3bBbP in the alternative complement pathway. The working point of CD59 resides in the reaction represented by a reference numeral (4) in FIG. 1. Different from CD46 and CD55, CD59 does not act on the C3 convertase but inhibits the conversion of C9 to ZC5b-9, which is a final step of the complement pathway.
These three mbCRPs can regulate respective steps of the complement pathway in the working mechanisms mentioned above. In addition, since these three proteins can be maintained with a high density at a rejection site due to the binding onto the membrane, they play an effective role to some extent in suppressing the complement pathway.
However, these mbCRPs have the following problems.
First, it is difficult to express CD46 in a transgenic animal abundantly. In addition, CD46 has a drawback in that it is poor in complement suppressing ability when bounded onto the membrane, compared to the other two factors.
It is known that CD59 regulates complement at the end of a cascade, so that C4, C3, and C5 present in the middle of the cascade are activated to generate anaphylatoxins, C4a, C3a and C5a, respectively, which damage a graft.
On the other hand, even if CD55, which is the most promising regulatory factor, is employed, the reactions up to C4 take place, with the result that C4a is generated and C4b is deposited on a graft.
The present invention has been made to overcome the aforementioned problems. An object of the present invention is to provide an mbCRP capable of effectively repressing a complement pathway and completely inhibiting generation of intermediates damaging a transplanted tissue in the complement pathway.
To be more specific, the present invention provides a membrane-bound C1 inhibitor comprising a protein containing a functional domain of a water-soluble C1 inhibitor and an anchor molecule attached to an end and/or an interior portion of the protein.
Additional objects and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out hereinafter.